1. Field of the Invention
The present invention relates to microbial industries. More specifically, the present invention relates to a method of producing L-lysine by fermentation, and a microorganism used for the production method.
2. Brief Description of the Related Art
L-amino acids such as L-lysine, L-glutamic acid, L-threonine, L-leucine, L-isoleucine, L-valine, and L-phenylalanine are industrially produced by fermentation using microorganisms that belong to the genus Brevibacterium, Corynebacterium, Bacillus, Escherichia, Streptomyces, Pseudomonas, Arthrobacter, Serratia, Penicillium, Candida or the like. Strains isolated from nature, or artificial mutants thereof, have been used to improve the productivity of these microorganisms. Moreover, various technologies have been disclosed to increase the ability to produce L-amino acids, such as using recombinant DNA technology to enhance L-amino acid biosynthetic enzyme activity.
As for bacteria belonging to the genus Escherichia, for example, a method of producing L-lysine by using a strain in which dihydrodipicolinate synthase activity is enhanced has been disclosed (JP 56-018596 A and U.S. Pat. No. 4,346,170).
Furthermore, a method of producing L-lysine is disclosed in WO 95/16042 (U.S. Pat. No. 6,040,160), wherein a strain is transformed with a plasmid containing the following: a DNA encoding Escherichia dihydrodipicolinate synthase having a mutation to desensitize feedback inhibition by L-lysine, a DNA encoding aspartokinase that is desensitized to feedback inhibition by L-lysine, a DNA encoding dihydrodipicolinate reductase, and a DNA encoding diaminopimelate dehydrogenase derived from a coryneform bacterium.
Furthermore, production of L-lysine has been reported to be improved by enhancing an activity of aspartate-semialdehyde dehydrogenase or phosphoenolpyruvate carboxylase, and activities of nicotinamide adenine dinucleotide transhydrogenase or aspartase in an Escherichia bacterium having a DNA encoding a Escherichia dihydrodipicolinate synthase that has a mutation desensitizing feedback inhibition to L-lysine, a DNA encoding an aspartokinase which is desensitized to feedback inhibition to L-lysine, a DNA encoding a dihydrodipicolinate reductase, and a DNA encoding a diaminopimelate dehydrogenase derived from a coryneform bacterium (WO 01/53459 (EP 1,253,195 A)).
As for coryneform bacteria, WO 95/11985 (U.S. Pat. No. 5,830,716) discloses that L-lysine productivity can be improved by increasing the intracellular activity of nicotinamide dinucleotide transhydrogenase.
Furthermore, a method of producing L-lysine using a strain in which phosphoenolpyruvate carboxylase activity is enhanced and a method of producing L-lysine using a strain in which aspartate-semialdehyde dehydrogenase activity is enhanced are disclosed in JP 60-87788 A and JP 06-102028 B, respectively.
It has also been reported that by introducing a dihydrodipicolinate synthase gene in addition to a mutant aspartokinase gene which is desensitized to feedback inhibition, L-lysine productivity can be improved (Applied and Environmental Microboiology 57(6), 1746-1752 (1991)).
Moreover, a method of significantly improving the yield of L-lysine without suppressing growth by introducing into a coryneform bacterium a plurality of L-lysine biosynthetic genes, specifically, mutant aspartokinase, dihydrodipicolinate reductase, dihydrodipicolinate synthase, diaminopimelate decarboxylase, and diaminopimelate dehydrogenase in combination, has also been disclosed (WO 96/40934 (EP 841,395, US 2003-0054506A)).
Also, in recent years, the LysE protein and gene encoding it were discovered. The LysE protein functions to export L-lysine specifically to the outside of a microorganism. Also, it has been reported that by increasing the LysE gene in a cell, the L-lysine-producing ability of the cell is also increased (Molecular Microbiology 22:815-826 (1996) or WO 97/23597 (U.S. Pat. No. 6,858,406)).
It has also been reported that the production of some L-amino acids can be improved by increasing the expression of the proteins responsible for amino acid export in Escherichia coli (JP 2000-189180 A (EP 1,016,710 A)). For example, it has been reported that production of cystine, cysteine, and the like can be improved by enhancing the expression of the ORF306 gene in Escherichia coli (EP 885,962 B).
Production of L-amino acids has been considerably increased by breeding of microorganisms and improving production methods as mentioned above. However, in order to respond to further increases in demand in the future, development of more efficient and less expensive methods is still desirable.
Methanol is known to be a raw material for fermentation and is available in large quantities at a low cost. Methods of producing L-amino acids by fermentation using methanol are known, and include those using microorganisms that belong to the genus Achromobacter or Pseudomonas (JP 45-25273 A), Protaminobacter (JP 49-125590 B), Protaminobacter or Methanomonas (JP 50-25790 A), Microcyclus (JP 52-18886 A), Methylobacillus (JP 04-91793 A), Bacillus (JP 03-505284 A), and the like.
Methods of producing L-amino acids by breeding bacteria belonging to the genus Methylophilus using artificial mutagenesis and recombinant DNA technology have been developed, resulting in enhancing the activities of dihydrodipicolinate synthase and aspartokinase (WO 00/61723 (EP 1,188,822 A, U.S. Pat. No. 7,223,572)). It was recognized that a big hurdle in the amino acid production by fermentation of methanol using a methanol-assimilating bacterium is the amino acid export process. In order to solve the problems, a mutant was developed with L-lysine-exporting activity in a methanol-assimilating bacterium from a LysE protein that is isolated from a bacterium belonging to the genus Corynebacterium and involved in export of L-lysine. It was shown that L-lysine can be efficiently produced by: breeding bacteria belonging to the genus Methylophilus using artificial mutagenesis, a recombinant DNA technology, and a modified L-lysine exporter, and the inventors have developed a method of producing an L-amino acid using a bacterium belonging to the genus Methylophilus (WO 00/61723 (EP 1,188,822 A, U.S. Pat. No. 7,223,572) or JP 2004-166594 A (US 2005-003495 A)).
However, for the production of L-amino acids by fermentation of methanol using a methanol-assimilating bacterium, there has been no report of significant improvements in L-lysine yield by enhancing expression of an L-lysine export gene in combination with a plurality of L-lysine biosynthetic genes.
It is known that enhancement of either ddh or lysA in a coryneform bacterium can improve the amount of produced Lys. It has also been disclosed that enhancement of ddh and lysA in combination can improve the Lys production rate and the amount of produced Lys.
However, enhancing either of these genes in a bacterium belonging to the genus Methylophilus is not known to increase, and may slightly decrease the amount of L-lysine that is produced.